This invention relates to nucleic acid and amino acid sequences of a novel phosphatidylinositol synthase and to the use of these sequences in the diagnosis, prevention, and treatment of diseases associated with abnormal phosphatidylinositol synthesis and metabolism.
Phosphatidylinositol (PI) is an essential lipid component of cell membranes. PI synthase (PIS) catalyzes the final step in PI biosynthesis, the transfer of myo-inositol to cytosine diphosphate (CDP)-diacylglycerol with liberation of cytosine monophosphate (CMP). PIS has been purified to varying levels of homogeneity from the yeast Saccharomyces cerevisiae (Fischl, A. S. et al. (1986) J. Biol. Chem. 261:3178-83) and from mammalian tissues including rat liver and human placenta (Monaco, M. E. et al. (1994) Biochem J. 304:301-305; Antonsson, B. E. (1994) Biochem J. 297:517-522). In mammalian tissues, the membrane-associated enzyme is localized primarily in the endoplasmic reticulum and the Golgi. The gene encoding PIS has been cloned from yeast and from rat brain (Nikawa, J. et al. (1987) J. Biol. Chem. 262:4876-4881; Tanaka, S. et al. (1996) FEBS Letts. 393:89-92).
A unique characteristic of PI over other cellular phospholipids is that the inositol headgroup can be further phosphorylated. Phosphorylation of PI at one or a combination of the 3xe2x80x2, 4xe2x80x2 and 5xe2x80x2 positions of the inositol ring generates regulatory molecules involved in a wide variety of cellular processes. For example, PI-4,5-bisphosphate (PI-4,5-P2) is hydrolyzed to the second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to agonist stimulation of various hormone and neurotransmitter receptors (reviewed in Berridge M. J. (1987) Ann. Rev. Biochem. 56:159-193). These second messengers are involved in the regulation of cell metabolism, contraction, secretion and proliferation. Other phosphorylated derivatives of PI, collectively known as phosphoinositides, are regulators of processes such as vesicular transport, cytoskeleton formation and maintenance, and cell growth (De Camilli, P., et al. (1996) Science 271:1533-1539).
Some diseases and conditions with which abnormal PI synthesis or metabolism have been associated include Down""s syndrome (DS), Lowe oculocerebrorenal syndrome (OCRL), and diabetic neuropathy. Patients with DS suffer from numerous functional disorders including mental retardation, microcephaly, cardiovascular malformations, immunological disorders, leukemia, and cataracts. Altered neuroelectrical properties, decreased Na+,K+-ATPase activity and plasma membrane abnormalities are also present. The concentration of myo-inositol in the cerebrospinal fluid (CSF) of DS patients is significantly elevated over age-matched controls, yet in DS plasma it is increased only slightly over normal plasma. The source of the increase in CSF myo-inositol, be it increased transport from plasma, increased de novo synthesis in the brain, or decreased catabolism, was not determined in this study (Shetty, H. U. et al. (1995) J. Clin. Invest. 95:542-546).
OCRL is a disorder involving several organ systems, including the eyes, nervous system, and kidneys, and characterized by congenital cataracts, renal tubular dysfunction and neurological deficits. OCRL is proposed to arise from an inborn error of inositol phosphate metabolism (Attree, O. et al. (1992) Nature 358:239-242). The primary defect in OCRL is a deficiency of a Golgi PI-4,5-P2 phosphatase (Suchy, S. F. et al. (1995) Hum. Mol. Genet. 4:2245-2250). The regulation of PI-4,5-P2 levels appear to be important in the modulation of Golgi vesicular transport. Suchy et al. suggest that disregulation of PI-4,5-P2 affects Golgi vesicular transport, which leads to developmental defects in the lens and abnormal renal and neurological function.
Diabetic neuropathy, a segmental demyelination of the peripheral nerves, is a complication of both insulin dependent and non-insulin dependent diabetes mellitus. The most common form of diabetic neuropathy first affects the sensory nerves of the lower limbs. In diabetic rats, a considerable decline in the incorporation of myo-inositol into PI in the sciatic nerve was observed (Bell, M. E. et al. (1985) J. Neurochem. 45:465-469). A metabolic pool of myo-inositol preferentially used for PI synthesis is depleted in rat diabetic peripheral nerve (Zhu, X. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9818-9822). The diminished PI synthesis and turnover observed in diabetic rat nerves has been causally linked to reduced Na+, K+-ATPase activity in theses nerves (Zhu, supra).
PI also plays an important role in protein membrane anchoring. A wide range of cell-surface proteins, including enzymes, coat proteins, and adhesion molecules, are attached to cell membranes via glycosyl-PI (GPI) anchors. GPI anchors are also proposed to function in intracellular sorting and transmembrane signaling. GPI anchors of plasma membrane proteins are present in eukaryotes from protozoa and fungi to vertebrates (Doering, T. L. et al. (1990) J. Biol. Chem. 265:611-614).
The initial step in GPI anchor formation is the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to PI. Whether this glycosylation step is specific for a certain class of PI is not known (Doering, supra). The inositol-fatty acyl sidechains of GPI anchors are quite variable, and are in general different from the cellular pool of PI phospholipids (McConville, M. J. et al. (1993) Biochem. J. 294:305-324). Fatty acid replacement or remodeling has been shown to occur subsequent to GPI anchor synthesis but, in some instances, PI molecules containing specific fatty acid sidechains may be the preferred GPI anchor precursors (Doering, supra).
A human disorder linked to defective GPI anchor biosynthesis is paroxysmal nocturnal hemoglobinuria (PNH). PNH is an acquired blood disorder which results from a somatic mutation in hematopoietic stem cells. Red blood cells arising from the PNH stem cells are highly sensitive to complement-mediated lysis and are prone to intravascular hemolysis. PNH may evolve to aplastic anemia or to acute leukemia. PNH cells are deficient in membrane surface GPI-linked proteins, which results from defects in GPI anchor biosynthesis (Hillmen, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:5272-5276).
Protozoan parasites cause widespread and debilitating diseases in humans and domestic livestock throughout the tropics. Examples of these diseases include malaria (caused by Plasmodium falciparum), African sleeping sickness and the cattle disease nagana (caused by Trypanosoma brucei), Chagas"" disease (caused by Trypanosoma cruzi), and kala azar, espundia, and Oriental sore (caused by Leishmania sp.). There are no vaccines against these diseases, and most of the available drug treatments are toxic and/or ineffective. Recently, drug resistant Plasmodium have placed malaria back into this category. The World Health Organization has identified the development of new and safer treatments for these diseases as a major priority.
Carbohydrate structures fixed into the parasite membrane by GPI membrane anchors play vital roles in the life cycles of these parasites. The use of GPI anchors is far more pronounced in parasites than in animal cells; in fact, GPI-anchored proteins dominate the molecular architecture of the parasite cell surface. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of Leishmania, are known to be essential for parasite survival and infectivity (Ferguson, M. A. et al. (1994) Parasitology 108: S45-54). Functional differences between some GPI-anchor biosynthetic enzymes of protozoan parasites compared to mammals have been noted (Guther, M. L. et al. (1994) J. Biol. Chem. 269:18694-18701).
Fungal infections are also major health problems, especially among immunocompromised individuals. For instance, patients are immunosuppressed to prevent the rejection of transplants and to treat neoplastic and inflammatory diseases. In addition, some infections, most notably that caused by human immunodeficiency virus (HIV), immunocompromise the host. Infectious agents that coexist peacefully with immunocompetent hosts wreak havoc in those who lack a complete immune system. Pulmonary infections by fungi such as Histoplasma sp. and Coccidioides immitis may be fatal in immunocompromised individuals, young children, or elderly patients. Patients with diabetes mellitus or hematologic malignancy, or those receiving broad-spectrum antibiotics or high doses of adrenal corticosteroids, are especially susceptible to tissue invasion by Candida. Aspergillus is another widespread fungus which does not commonly cause disease except in immunocompromised patients.
The discovery of polynucleotides encoding phosphatidylinositol synthase, and the molecules themselves, provides a means to investigate phosphatidylinositol synthesis and metabolism in normal and diseased cells. Discovery of molecules related to phosphatidylinositol synthase satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment or prevention of neurological, renal, ocular, or other systemic dysfunction in diseases associated with abnormal PI metabolism, including diabetic neuropathy, Down""s syndrome, OCRL, and diseases associated with abnormal glycosyl-PI anchor biosynthesis such as PNH. Knowledge and expression of sequences encoding human phosphatidylinositol synthase is also useful for developing therapeutic agents to prevent or treat diseases associated with fungal and parasitic infections.
The present invention features a novel phosphatidylinositol synthase hereinafter designated PISH and characterized as having similarity to phosphatidylinositol synthase from rat and from yeast.
Accordingly, the invention features a substantially purified PISH having the amino acid sequence shown in SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode PISH. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode PISH. The present invention also features antibodies which bind specifically to PISH, and pharmaceutical compositions comprising substantially purified PISH. The invention also features the use of agonists and antagonists of PISH.